Sandwich immunoassay utilizing a separation specific binding substance

An immunoassay method for determining the amount of each of a plurality of polyvalent ligands in a liquid test sample, each of said ligands being an antibody or a substance capable of specific binding to an antibody, said method comprising the steps of;

• (a) providing an insoluble phase to which is bound at least one separation specific binding substance which does not bind any of said ligands, wherein each said separation specific binding substance is an antibody which specifically binds the carborane moiety of a hapten comprising a carborane moiety, said hapten being conjugated to a capture specific binding substance;
• (b) incubating said insoluble phase with
• (1) said liquid test sample;
• (2) for each said ligand, a capture specific binding substance which specifically binds said ligand, and which is itself conjugated to a hapten comprising a carborane moiety which is specifically bound by at least one of said separation specific binding substances;
• and
• (3) for each said ligand, a probe specific binding substance which specifically binds said ligand at a site which is accessible when said ligand is bound to said capture specific binding substance, each said probe specific binding substance being not substantially bound by said separation specific binding substance, and each being separately detectable by at least one detection procedure;
• (c) separating said insoluble phase, after said incubation, from resultant liquid phase containing unbound reagents and components of said test sample;
• and
• (d) determining, by means of said detection procedure, the amount of each said detectable probe which is bound to said separated insoluble phase, or the amount of each said detectable probe remaining in said resultant liquid phase, said determination being related to the amount of said ligand in said test sample.

An immunoassay kit, suitable for determining the amount of a polyvalent ligand in a liquid test sample, comprising:

• (a) an insoluble phase to which is bound a separation specific binding substance which does not bind said ligand, wherein said separation specific binding substance is an antibody which specifically binds the carborane moiety of a hapten comprising a carborane moiety, said hapten being conjugated to a capture specific binding substance;
• (b) a capture specific binding substance which specifically binds said ligand, and which is itself conjugated to said hapten comprising said carborane moiety which is specifically bound by said separation specific binding substance;
• and
• (c) a probe specific binding substance which specifically binds said ligand at a site which is accessible when said ligand is bound to said capture specific binding substance, said probe specific binding substance being not substantially bound by said separation specific binding substance, and being detectable by at least one detection procedure.

An immunoassay kit, suitable for determining the amount of each of a plurality of polyvalent ligands in a liquid test sample, each of said ligands being an antibody or a substance capable of specific binding to an antibody, said kit comprising;

• (a) an insoluble phase to which is bound at least one separation specific binding substance which does not bind any of said ligands, wherein each said separation specific binding substance is an antibody which specifically binds the carborane moiety of a hapten comprising a carborane moiety, said hapten being conjugated to a capture specific binding substance;
• (b) for each said ligand, a capture specific binding substance which specifically binds said ligand, and which is itself conjugated to a hapten comprising a carborane moiety which is specifically bound by at least one of said separation specific binding substances;
• and
• (c) for each said ligand, a probe specific binding substance which specifically binds said ligand at a site which is accessible when said ligand is bound to said capture specific binding substance, each said probe specific binding substance being not substantially bound by said separation specific binding substance, and each being separately detectable by at least one detection procedure.